1. Field of the Invention
The present invention relates generally to the analysis of fluid samples and, more particularly, to the analysis of fluid samples in a photometric flow cell. The invention is particularly suited for use in liquid chromatography in which a stream of successive fluid sample fractions eluted from a chromatographic column is flowed through a photometric cell for analysis.
2. Description of the Prior Art
Numerous photometric flow cells have been developed for measuring optical properties of fluid samples flowed through cells. Typically, a flow passageway of the cell is bounded at opposite ends by optically transparent windows which permit a light beam to be directed into one end, along the length of, and out the other end of the flow cell passageway. Conduits connected to entrance and exit openings of opposite ends of the passageway provide for introduction of fluid at one end and for removal of fluid at the other end of the passageway. A light detector intercepts light exiting the flow cell to provide a measure (usually absorbance) of the effect of the sample on the light. From this measurement quantitative and qualitative information regarding the sample is derived.
Flow cells of the foregoing nature can be grouped generally as either static or dynamic. In a static flow cell, the photometric light measurement is performed with the fluid sample at rest in the flow cell passageway. In a dynamic flow cell, on the other hand, the photometric measurement is made as the sample is flowing through the passageway. As a result, dynamic flow cells are particularly prone to measurement errors from turbulence or other disruptions in the flowing sample.
In high-pressure liquid chromatography (HPLC) sample analysis in dynamic flow cells is complicated by the fact that measurements are made on flowing samples which are minute in volume and which are flowed through the cell at high pressure. In this respect, HPLC sample fractions (carried in a solvent matrix) are typically between 30 .mu.l-1.0 ml in volume and are flowed at rates between 0.5-2.0 ml/min at pressures between 100-300 atmospheres through conduits as small as 0.25 mm inside diameter. In order to minimize spreading of sample within the solvent matrix, the sample volume is minimized and the conduit connecting the chromatographic column and the flow cell is configured to have minimum length (i.e. several inches) and interior diameter. For such applications, a representative flow cell is typically dimensioned with a flow passageway 1.0 cm in length and between 1.0-2.0 mm inside diameter to define a total flow passage volume of about 10-20 .mu.l.
With such dimensional restrictions as above, it is imperative in HPLC applications that the flow of sample fractions through the flow cell approach plug flow, i.e. flow such that each minute sample fraction is retained as a discrete fluid segment all portions of which travel at the same velocity with a flat front and for which mixing between successive segments is avoided. Unfortunately, ideal plug flow is difficult to attain in practice. Such is caused in part by the geometry of the flow cell passageway and the conduits which deliver and remove the sample fractions. In this regard, an abrupt and turbulent flow transition is created by the relatively narrow diameter conduit opening into one end of the wider diameter flow passageway. Fluid flowed through the conduit into and through the entrance opening of the passageway follows the path of least resistance through only one side of the passageway opening, and turbulence is created in the entering fluid as it then spreads out across the flow passageway. Because of such turbulence, the flow cell exhibits several problems in operation including: (1) instability in the optical baseline between measurement of successive fluid fractions, particularly fluids of differing indices of refraction, (2) mixing between successive fractions, (3) formation of air bubbles in the flow cell passageway, and (4) formation of dead pockets or corners in the flow passageway which require relatively large volumes of fluid (e.g. 2-3 cell volumes) to totally sweep or flush each sample from the passageway. A turbulent transition at the exit opening of the flow passageway can cause similar problems.
Accordingly, a need exists for a flow cell adapted for dynamic optical measurement of continuously flowing fluid sample without the drawbacks of the prior art. The present invention meets these needs.